Crsytallization and Structural Analysis of Archaeal Homolog of Ski-2 type RNA helicase from the Hyperthermophilic Archaeon Pyrococcus horikoshii

Department of Bioscience and Biotechnology, Kyushu University* Graduate School of Systems Life Sciences, Kyushu University, Japan**
â—‹Takashi Nakashima* Xiaodong Zhang** Yoshimitsu Kakuta* Makoto Kimura*

The SKI (superkiller) genes were first identified from mutations that cause overexpression of a killer toxin encoded by the endogenous double-stranded RNA. Subsequent studies revealed that the products (Ski2p, Ski3p, and Ski8p) of SKI2, SKI3, and SKI8 genes, respectively, are necessary for the 3'- to -5' mRNA degradation and repression of translation of non- polyadenylated RNA in addition to their antiviral activities. Furthermore, it was found that Ski2p, Ski3p, and Ski8p form the Ski complex and play an essential role in cytoplasmic mRNA turnover as cofactors for the exosome in yeast. However, the specific role of the Ski complex in 3' mRNA decay still remains elusive.
Ski2p is a putative RNA helicase with DEVH motif and conserved among eukaryote and archaea. We found that the gene PH1280 product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii shows sequence homology to the yeast Ski2p: the N-terminal domain of Ph1280p shares 36% identical amino acid with the putative catalytic DEVH helicase domain of the yeast Ski2p. In order to elucidate the molecular mechanism of the RNA helicase in the Ski complex, we overproduced Ph1280p in Escherichia coli cells and crystallized hanging drop vapor diffusion method.
The crystals diffract up to 3.6 Å resolution and belong to the space group C2221 with unit cell parameters a = 283.3 Å, b = 490.0 Å, c=76.2 Å. The crystal contains molecule in the asymmetric unit, with a solvent content of 54% and a Vm value of 2.7 Å3/Da. The MAD data collection was performed with two wavelengths near the absorption peak of selenium, and the model building of the Ph1280p is now in progress.